Research ArticleIdentification and Phylogenetic Analysis ofthe Complete Chloroplast Genomes of Three EphedraHerbs Containing Ephedrine

Xinlian Chen ,1 Yingxian Cui ,1 Liping Nie ,1 Haoyu Hu ,2 Zhichao Xu ,1

Wei Sun ,2 Ting Gao ,3 Jingyuan Song ,1 and Hui Yao 1

1Engineering Research Center of Tradition Chinese Medicine Resource, Ministry of Education, Institute ofMedicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100193, China2Institute of Chinese Materia Medica, China Academy of Chinese Medicinal Sciences, Beijing 100700, China3Key Laboratory of Plant Biotechnology in Universities of Shandong Province, College of Life Sciences, Qingdao Agricultural University,Qingdao 266109, China

Correspondence should be addressed to Hui Yao; scauyaoh@sina.com

Received 13 November 2018; Accepted 3 February 2019; Published 3 March 2019

Academic Editor: Pengjun Shi

Copyright © 2019 Xinlian Chen et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Ephedrae Herba and Ephedrae Radix et Rhizoma (Mahuang) have been used as Chinese herbal medicines. Ephedra plants mainlylive in deserts and have good governance of desertification. Despite their important medicinal and environmental protection value,dietary supplements containing ephedrine from Ephedra species may threaten the health of people. Morphological resemblanceamongst species causes difficulty in identifying the original species of Ephedra herbs. Chloroplast (CP) genome shows goodprospects in identification and phylogenetic analysis. This study introduced the structures of the CP genomes of three Ephedraspecies and analysed their phylogenetic relationships.Three complete CP genomes of Ephedra showed four-part annular structures,namely, two single-copy regions and two inverted repeat regions. The entire CP genomes of three Ephedra species in terms ofsize were 109,550 bp (E. sinica), 109,667 bp (E. intermedia), and 109,558 bp (E. equisetina). Each CP genome of the three Ephedraspecies encoded 118 genes, including 73 protein-coding genes, 37 tRNA genes and 8 ribosomal RNA genes. Eleven high-variationregionswere screened throughmVISTA to be potential specificDNAbarcodes for identifyingEphedra species.Maximum likelihoodand maximum parsimony trees showed that CP genomes could be used to identify Ephedra species. The Ephedra species had aclose phylogenetic relationship withGnetum species andWelwitschia mirabilis.This research provided valuable information for theidentification and phylogenetic analysis of gymnosperms and drug safety of Ephedra.

1. Introduction

Ephedrae Herba (Mahuang), as a Chinese herbal medicine,is the dry grassy stem of Ephedra sinica Stapf, E. intermediaSchrenk et C. A. Mey. or E. equisetina Bge. It is used forperspiratory, antitussive, antipyretic, and anti-inflammatorypurposes [1]. It has also been utilised for more than 2500years [2, 3]. It is also applied to Kampo medicine in Japan[4]. Similarly, the Chinese herbalmedicine Ephedrae Radix etRhizoma comes from the dry roots and rhizomes of E. sinicaor E. intermedia [1]. It is an antiperspirant and used for spon-taneous sweating and night sweat. Ephedra (Ephedraceae)

belongs to Gymnospermae and comprises approximately 40known species.They are distributed in arid and desert regionsranging from Asia and Southeastern Europe to NorthernAfrica and America [5]. Their unique habitat indicates thatthey have powerful resistance to drought, cold, and sandburial and are commonly used as a sand binder [5, 6].

Ephedrae Herba contains ephedrine [7], pseudoephedrine[8], norpseudoephedrine [9], and methylephedrine [10, 11],which are abundant in the three Ephedra plants and havepharmaceutically active anodyne and antifebrile effects [12].The highest alkaloid content is found inE. equisetina followedby E. sinica and E. intermedia. E. sinica [5, 13] and E.

HindawiBioMed Research InternationalVolume 2019, Article ID 5921725, 10 pageshttps://doi.org/10.1155/2019/5921725

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equisetinamainly consist of ephedrine, whereas E. intermediamainly contains pseudoephedrine [13–15]. In some instances,Ephedra-based products are used as bronchodilators intraditional Asian medicines [16]. Since the 20th century,dietary supplements containing ephedra alkaloids have beenwidely promoted and used in America because of theireffects on weight loss and energy increase. However, thesesupplements may threaten the health of people [17]. TheFood and Drug Administration prohibited the sale of dietarysupplements containing Ephedra spp. or ephedrine alkaloidsin April 2004 [18]. Using Ephedra or ephedrine and caffeineis associated with an increased risk of psychiatric, autonomic,or gastrointestinal symptoms and heart palpitations [19].

Few contrasting morphological characters are observedwhen Ephedra species do not bear flowers or seeds, therebycausing difficulty in identifying the original species ofEphedra Herb. This genus has also been systematically stud-ied [20]. Different Ephedra species, habitats, and pickingtimes can be distinguished by diffuse reflectance Fouriertransform near infrared spectroscopy [21]. Ephedra specieshas been identified, and their phylogenetic relationship hasbeen reconstructed through chloroplast and nuclear DNAsequences. Results have been applied to identify crude drugsobtained in the Chinese market [20, 22, 23]. ITS2 sequenceshows a sufficient resolution amongst EphedraeHerba and itsclosely related species but fails to distinguish amongst threeoriginal Ephedrae Herba species (E. sinica, E. intermedia, andE. equisetina) [24].

Chloroplast plays an important role in photosynthesis,transcription, or translation [25]. As one of the three genomesof plants, CP genome shows good potential for species iden-tification and phylogenetic reconstruction [26–28]. Studieshave aimed to use the entireCP genome as a super barcode forspecies identification [29–31]. In this study, the structures ofthe CP genomes of the three Ephedra species were introducedin the Chinese Pharmacopoeia, and the identification abilityof the CP genomes on this genus was analysed. This studyprovided invaluable information for studies on gymnospermidentification and phylogenetic analysis.

2. Materials and Methods

2.1. DNA Sources. Fresh E. intermedia stems were collectedfrom Altay Prefecture, Xinjiang Uygur Autonomous Region,China. Fresh E. sinica and E. equisetina stems were obtainedfrom BeijingMedicinal Plant Garden,The Institute ofMedic-inal Plant Development (IMPLAD), Chinese Academy ofMedical Sciences.These three species were identified by Prof.Yulin Lin from IMPLAD. Voucher specimens were depositedin the herbarium at IMPLAD.

2.2. DNA Sequencing, Assembly and Annotation. Total DNAwas extracted with a DNeasy Plant Mini Kit (Qiagen Co.,Germany). Shotgun libraries with insert sizes of 500 bp werebuilt. Total DNA was sequenced in Illumina HiSeq X. Thelibraries were sequenced on an Illumina HiSeq X platformto produce 150 bp paired-end reads. Low-quality reads andadapters were filtered from the raw data by using Trim-momatic [32]. Then, the remaining clean reads were used

to assemble the CP genome sequences. The CP sequencesof all plants downloaded from the National Center forBiotechnology Information (NCBI) constituted the referencedatabase. Subsequently, the clean sequences were mapped tothe database, and the mapped reads were extracted on thebasis of coverage and similarity. The extracted reads wereassembled into contigs by using SOAPdenovo2 [33]. Thescaffold of the CP genome was constructed using SSPACE[34], and gaps were filled using GapFiller [35]. The accuracyof the assembly of the four boundaries, namely, large single-copy (LSC), small single-copy (SSC), and inverted repeat (IRaand IRb) regions, was verified by amplicons obtained fromspecific polymerase chain reaction primers (Table S1).TheCPgenomes of the three Ephedra species were initially annotatedusing the online programs Dual Organellar GenoMe Anno-tator [36] and CPGAVAS [37] and then manually corrected.The assembled complete CP genome sequences of the threespecies were submitted to NCBI with the accession numbersMH161420 (E. equisetina), MH161421 (E. intermedia), andMH161422 (E. sinica).

2.3. Genome Analysis. tRNA genes were identified withtRNAscan-SE [38]. CP genome maps were generated usingOrganellar Genome DRAW (OGDRAW) v1.2 [39] and thenmanually corrected. The GC content was calculated usingMEGA 6.0 [40]. REPuter (University of Bielefeld, Bielefeld,Germany) [41] was employed to identify the size and locationof repeat sequences in the CP genomes of the three Ephedraspecies. Simple sequence repeats (SSRs) were detected withMISA (http://pgrc.ipk-gatersleben.de/misa/). All of therepeated sequences were manually verified, and excess datawere removed.The distribution of codon usage was estimatedusing MEGA 6.0 [40]. All these methods were also used inidentification of Ligularia herbs using complete CP genome(https://www.frontiersin.org/articles/10.3389/fphar.2018.00695/full) [31].

2.4. Phylogenetic Analysis. The mVISTA [42] was used tocompare the three Ephedra species and two publishedEphedra species with E. intermedia as a reference genome.Thenucleotide diversity of theCP genomewas analysed usingthe sliding window method implemented in DnaSP v5.10[43]. The step size was set to 200 bp with a window lengthof 800 bp. A phylogenetic tree with Selaginella uncinataand Equisetum arvense as outgroups was constructed on thebasis ofmaximum likelihood (ML) andmaximumparsimony(MP) analysis inMEGA6.0.Thedetails of the selected speciesexcluding the three Ephedra species are presented in Table S2.

3. Results and Discussion

3.1. Molecular Features of the CP Genomes of Three EphedraSpecies. Three complete CP genomes of Ephedra showedfour-part annular structures, namely, an LSC, an SSC, andtwo inverted repeat (IR) regions similar to most land plants(Figure 1) [44]. The lengths of the whole CP genomes were109,667 bp (E. intermedia), 109,550 bp (E. sinica), and 109,558bp (E. equisetina).The lengths of the LSC regions were 59,936bp (E. intermedia), 59,961 bp (E. sinica), and 59,976 bp (E.

BioMed Research International 3

polycistronic transcriptsorigin of replicationribosomal RNAs

transfer RNAs

ORFshypothetical chloroplast reading frames (ycf)other genes

clpP, matK

ribosomal proteins (LSU)

ribosomal proteins (SSU)

RNA polymerase

RubisCO large subunit

NADH dehydrogenase

ATP synthase

cytochrome b/f complex

photosystem II

photosystem I

ycf2

ycf2

ycf1

rpoC

2

rpoB

rrn23

rrn23

rpoC

1*

trnK-UUU*

psaA

psaB

ycf3*

matK

chlB

psbB

atpA

rrn16

rrn16

atpB

rbcL

psbC

chlN

chlN

rpl2*

petB*

*Fpt

a

psbD

psbA

petD*

rpl16*

petA

ccsA

chlL

chlL

trnA-U

GC*

trnA-UGC*trnI-GAU*

trnI-G

AU*

rpoA

cemA

atpI

rps12*

rps12*

rps2

clpP

rps3

rps4

ycf4

rps7

rps7

rpl22

atpE

rps11

rps8

trnL-UAA*

rpl14

rpl20

rps19

rps14

rps18

psbE

psaC

atpH

infA

rps1

5

rps15

psbHpsbZ

psbK

rpl33

rrn5

rrn5

psbN

psaJ

psbJpsbF

psbI

psbL

psbT

rpl36

rps12*

petG

psaIpsbM

rrn4.5

rrn4.5

petL

petN

trnS-GCU

trnS-G

GA

trnS-UGA

trnY-GUA

trnL-CAA

trnL-

UAG

trnL-CAA

trnP-UGG

trnH-GUG

trnN

-GU

U

trnI-CAUtrnI-CAU

trnR-ACG

trnR-

ACG

trnW-CCA

trnR-CCG

trnfM-CAU

trnD-G

UC

trnH-GUG

trnN-GU

U

trnT-UG

U

trnE-UU

C

trnM-CAU

trnF-GAA

trnQ-U

UG

trnV-GAC

trnV-G

AC

trnT-GGU

trnR-UCU

trnC-G

CA

trnG-UCC

Ephedra intermedia 109,667 bp

IRAIR B

S S C

LSC

Ephedra sinica

Ephedra equisetina

109,550 bp

109,558 bp

Chloroplast Genome

Figure 1: Gene maps of the complete CP genomes of the three Ephedra species. Genes on the inside of the circle are transcribed clockwise,while those outside are transcribed counter clockwise.The darker gray in the inner circle corresponds to GC content, whereas the lighter graycorresponds to AT content.

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Table 1: Statistics for assembly of the three CP genomes and two published of Ephedra species.

Latin name E. intermedia E. sinica E. equisetina E. equisetinaNC 011954

E. foemineaNC 029347

Gene size (bp) 109,667 109,550 109,558 109,518 109,584LSC (bp) 59,936 59,961 59,976 59,906 60,027SSC (bp) 8,247 8,103 8,078 8,104 8,079IR (bp) 20,742 20,743 20,752 20,754 20,739GC Content (%) 36.6 36.7 36.6 36.7 36.7

Table 2: List of genes found in the three CP genomes of Ephedra species.

No. Group of genes Gene names Amount1 Photosystem I psaA, psaB, psaC, psaI, psaJ 5

2 Photosystem II psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL,psbM, psbN, psbT, psbZ 15

3 Cytochrome b/f complex petA, 𝑝𝑒𝑡𝐵∗, 𝑝𝑒𝑡𝐷∗, petG, petL, petN 64 ATP synthase atpA, atpB, atpE, 𝑎𝑡𝑝𝐹∗, atpH, atpI 65 RubisCO large subunit rbcL 16 RNA polymerase rpoA, rpoB, rpoC1∗, rpoC2 4

7 Ribosomal proteins (SSU) rps2, rps3, rps4, rps7∗(×2), rps8, rps11, rps12∗∗(×2), rps14,rps15(×2), rps18, rps19 14

8 Ribosomal proteins (LSU) rpl2∗, rpl14, rpl16∗, rpl20, rpl22, rpl33, rpl36 79 Photochlorophyllide reductase subunit B/L/N chlB, chlL(×2), chlN(×2) 510 Proteins of unknown function ycf1, ycf2(×2), ycf3∗∗ , ycf4 511 Transfer RNAs 37 tRNAs (6 contain an intron) 3712 Ribosomal RNAs rrn4.5(×2), rrn5(×2), rrn16(×2), rrn23(×2) 813 Other genes clpP,matK, ccsA, cemA, infA 5∗Gene contains one intron; ∗∗gene contains two introns; (×2) indicates the number of the repeat unit is 2.

equisetina). The lengths of the SSC regions were 8,247 bp (E.intermedia), 8,103 bp (E. sinica), and 8,078 bp (E. equisetina).The lengths of the IR regions were 20,742 bp (E. intermedia),20,743 bp (E. sinica), and 20,752 bp (E. equisetina). The CPgenomes of Ephedra were the most reduced and compactamongst the elucidated photosynthetic land plants, suchas Ginkgo biloba [26], Cycas [45], Gnetum [46, 47], andWelwitschia mirabilis [48]. The GC contents were 36.6% (E.intermedia and E. equisetina) and 36.7% (E. sinica), whichwere lower than those of some gymnosperms [46, 47, 49].TheGC contents of the IR and LSC regions of the three specieswere 42% and 34.2%, respectively. The GC contents of theSSC regions were 27.3% (E. intermedia), 27.8% (E. sinica),and 27.5% (E. equisetina). The sizes of the four regions andthe GC contents of the three Ephedra species were similar topreviously published E. equisetina [48] and E. foeminea [50](Table 1).

The CP genomes of the three Ephedra species each onlyhad 118 genes, including 73 protein-coding genes, 37 tRNAgenes and 8 rRNA genes. The CP genomes consisted ofthe coding regions from 73.87% (E. intermedia) to 73.93%(E. equisetina), which were greater than those of somegymnosperms, such asW.mirabilis [48] andGnetum [46, 47].The rates of noncoding regions, including intergenic spacers

and introns, varied from 26.07% (E. equisetina) to 26.13% (E.intermedia). The genes of the CP genomes contained in thethree Ephedra species are shown in Table 2. Furthermore, 18duplicated genes were found in the IR regions: 6 protein-coding genes (chlL, chlN, rps12, rps15, rps7, and ycf2), 8 tRNAgenes (trnA-UGC, trnH-GUG, trnI-CAU, trnI-GAU, trnL-CAA, trnN-GUU, trnR-ACG, and trnV-GAC), and 4 rRNAgenes (rrn16, rrn23, rrn4.5, and rrn5). psbA spanned the IRband LSC regions, and 13 genes (atpF, rps7, petB, petD, rpl16,rpl2, rpoC1, trnA-UGC, trnT-UGU, trnI-GAU, trnK-UUU,trnL-CAA, and trnV-GAC) consisting of only one intronwere observed. In addition, 2 genes containing two introns(rps12 and ycf3) were found. matK was located within theintron of trnK-UUU in the Ephedra CP genomes. No NADHdehydrogenase genes were observed in the three Ephedra CPgenomes.

The codon content of the 20 amino acid and stop codonsin all of the protein-coding genes of the CP genomes ofEphedra species are shown in Figure 2. The Relative Syn-onymous Codon Usage (RSCU) of the three Ephedra speciesis shown in Table S3. RSCU revealed a high proportion ofsynonymous codons having A and U in the third positionin Ephedra. Protein-coding genes comprised 22,999 codonsin E. sinica to 23,023 codons in E. intermedia. Codons

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0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

D L I M N V F P Q K S E T A Y H C R W G Stop

Figure 2: Codon content of 20 amino acid and stop codons in all protein-coding genes of the CP genomes of three Ephedra species. The order ofevery three columns is E. intermedia, E. sinica, and E. equisetina, respectively.

for leucine, isoleucine, and lysine were the most abundant,whereas those for cysteine, tryptophane, and methioninewere the least.

3.2. Repeat Sequences and SSRs. Significant differences wereobserved in the number distribution of long repeat sequencesamongst the three Ephedra species (Figure 3). Our resultsrevealed 4 complement repeats, 10 forward repeats, 14 palin-dromic repeats, and 11 reverse repeats in the CP genome ofE. intermedia. Furthermore, 1 complement repeat, 5 forwardrepeats, 7 palindromic repeats, and 6 reverse repeats werefound in the CP genome of E. sinica. In the CP genomeof E. equisetina, 2 complement repeats, 6 forward repeats,8 palindromic repeats, and 9 reverse repeats were present.SSRs (1–6 nucleotide repeats) were abundant in the threeEphedra CP genomes. SSRs can offer relevant informationfor the analysis of phylogenetic relationships and populationgenetics [51–53]. The sequences of SSRs contained an A orT base, resulting in AT richness of the CP genome [54].The distributions of SSRs in the three species were detected.Mononucleotide repeats A and Twere the twomost commontypes (Table S3). Few other types were observed. The MISAsoftware identified 55 (E. intermedia) to 62 (E. sinica) SSRs inthe three Ephedra CP genomes. Most SSRs were distributedin the LSC and SSC regions. Each species of Ephedra hadspecies-specific SSRs. E. intermedia and E. sinica had one andtwo mononucleotide C SSRs, respectively, which were not inE. equisetina. E. sinica and E. equisetina contained one andtwo dinucleotide TA SSRs, respectively, whichwere not foundin E. intermedia. Only E. sinica contained one tetranucleotideTTCT SSRs. Only E. equisetina contained one tetranucleotideCTAT SSRs. The mass variation in SSRs in the three EphedraCP genomes would offer invaluable resources for the markerdevelopment and population genetics of this genus.

0

2

4

6

8

10

12

14

F P R C F P R C F P R C

30-3940-4950-59

60-69≥70

E. intermedia E. sinica E. equisetina

Figure 3: Repeat sequences in three CP genomes. REPuter was usedto identify repeat sequences with length ≥ 30 bp and sequencesidentified≥ 90% in theCP genomes. F, P, R, andC indicate the repeattypes F (forward), P (palindrome), R (reverse), andC (complement).Repeats with different lengths are indicated in different colors.

3.3. Comparative Analysis of Ephedra CP Genomes. Theannotated genes of the three studied Ephedra species andthe two published Ephedra species were compared usingmVISTA [42]. mVISTA (Figure 4) revealed that the CP

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matK

psbA trnK

E. intermediaE. sinicaE. equisetinaE. equisetina(NC_011954)E. foeminea(NC_029347) 0k 3k

rps2 atpI atpFatpH

psbCtrnG

chlB trnT psbD psaBtrnS rps14psbZ trnfM

6k 9ktrnR psbI trnY

atpA trnS trnQ trnF atpE atpB

psbK trnE trnL trnM

rpoC1

psaA ycf3 trnS psbM trnC rpoB rpoC2 rps2rps4 petN

100%

50%100%

50%100%

50%100%

50%12k 15k 18k 21k 24k 27k

psbF trnW rps11

rbcL psaI cemA petA psbL petL trnP

clpP psbB psbT

psbH petD rpoA rpl36ycf4 psbJpsbEpetG psaJ rps12 -5end psbN petB infA

contiggeneexonUTRCNSmRNA

100%50%100%50%100%50%100%50%

30k 33k 36k 39k 42k 45k 48k 51k 54krpl22 rpl2

trnLrps12-3endinfA rpl14 rps3 rps19 trnI ycf2 rrn16 trnI trnA rrn23 rrn4.5 trnR chlL chlN rps15 ccsA

rps8 rpl16 trnH rps7 trnV rrn5 trnN psaC

100%50%100%50%100%50%100%50%

57k 60k 63k 66k 69k 72k 75k 78k 81kchlL

trnL ycf1 rps15 chlN trnN rrn5 rrn23 trnA trnI rrn16 trnV rps7 ycf2 trnH psbA

trnR rrn4.5 rps12-3endtrnL trnI

100%50%100%50%100%50%100%50%

84k 87k 90k 93k 96k 99k 102k 105k 108k

Figure 4: Sequence identity plot comparing five CP genomes with E. intermedia as a reference using mVISTA. Gray arrows and thick black linesabove the alignment indicate genes with their orientation and the position of the IRs, respectively. A cutoff of 70% identity was used for theplots, and the Y-scale represents the percent identity ranging from 50 to 100%.

genomes of the five Ephedra species showed similarity andconservatism. The divergence level of the noncoding regionswas higher than that of the coding regions. The divergencelevel of the single-copy regions was higher than that of theIR regions. Approximately 11 high-variation regions werefound inmVISTA, and they were distributed in the sequencesmainly in noncoding regions, including psbZ-trnG, petN-rpoB, trnR-trnM, psbJ-rpl20, clpP-psbB, rrn16-trnI, rps15-ccsA, ycf1-rps15, and trnV-rps12, and in two genes, namely, ycf3and rpl2. These sequences could provide potential informa-tion to identify Ephedra species. In addition, the boundariesof the four regions of the three Ephedra CP genomes werecompared in detail (Figure 5). In the junction positions,the sites of most genes in the border region were similar.However, ycf1 was located entirely on the left of the SSC-IRb boundary in E. intermedia, whereas 18 bp was located

in the IRb regions in ycf1 in E. sinica and E. equisetina. Theaverage nucleotide diversity (Pi) amongst the three Ephedraspecies was 0.00252 (Figure 6).Mutational hotspots with highPi values (>0.008) were located in the LSC and SSC regionsrather than in the IR regions.

3.4. Identification and Phylogenetic Analysis. Chloroplastgenome has important implications for phylogenetic studies[28, 55]. In addition to three Ephedra species, 16 specieswere chosen to construct ML and MP trees to identify thephylogenetic position of Ephedra species based on 53 com-mon protein-coding genes by using MEGA 6.0 (Figure 7).The 16 species were from Gymnospermae (14 species) andPteridophyta (Equisetum arvense and Selaginella uncinata asoutgroups). The alignment covered 52,722 bp. The resultsrevealed the same tree topology of ML and MP. Each species

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E. sinica

E. equisetina

E. intermedia

IRa SSC IRb59,961 bp 20,743 bp 8,103 bp 20,743 bp

IRa IRb59,976 bp 20,752 bp 8,078 bp 20,752 bp

LSC IRa SSC IRb LSCpsbArps15

59,936 bp 20,742 bp 8,247 bp 20,742 bp

LSC LSC

psbA

LSC SSC IR LSCpsbA

psaCrps15

rpl2trnI-CAU

rps15

ycf1

psaC

rps15rpl2trnI-CAU

ycf1

rps15 psaC

rps15trnI-CAU

rpl2

ycf1

trnI-CAU

trnI-CAU

51 bp

501 bp

501 bp

501 bp

51 bp

51 bp

80 bp

84 bp

84 bp

234 bp

217 bp

216 bp

trnI-CAU128 bp80 bp

66 bp

68 bp

Figure 5: Comparison of the borders of LSC, SSC, and IR regions amongst three CP genomes. Number above the gene features means thedistance between the ends of genes and the borders sites. These features are not to scale.

0.01 0.01

0.00833

0.0125

0.00833

0.01667

0.010830.00917

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0

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0 20000 40000 60000 80000 100000 120000

Pi

Pi

LSC IRaSSCIRb

Figure 6: Sliding window analysis of the whole CP genomes. Window length: 800 bp; step size: 200 bp. X-axis: position of the midpoint of awindow. Y-axis: nucleotide diversity of each window. Pi amongst three Ephedra species. Mutational hotspots and highly divergent loci weremarked.

of Gymnospermae and Pteridophyta clustered into a mono-phyletic group on the basis of topologic structure. Gym-nospermae species were divided into two branches (Clade Aand Clade B). Clade A was divided into two subbranches,namely, Clade A1 and Clade A2, with a bootstrap supportvalue of 100%. In Clade A, Clade A1 formed a stronglysupported monophyletic clade sister to Clade A2. Eachbranch of Ephedra species showed high support (bootstrapvalue ≥ 89%), indicating that the four Ephedra species couldbe identified. Two E. equisetina and E. intermedia clusteredinto amonophyletic clade, indicating their close phylogeneticrelationship. Clade A2 included Gnetum species and W.mirabilis, revealing a close phylogenetic relationship withEphedra. These data could be used for the identification,phylogenetic analysis, and population studies of Ephedraspecies.

4. Conclusions

In this study, the CP genomes of three Ephedra specieswere sequenced and analysed. The results revealed the basicstructures, conservation, and variability of the sequences.Eleven variation regions were screened to be potential DNAbarcodes for the identification of this genus.TheML andMPtrees indicated that the CP genomes could be used to identifyEphedra species. Ephedra species showed a close phylogeneticrelationship with Gnetum species and W. mirabilis. Thedata obtained in this study would be a helpful basis forfurther research involving the identification and phylogeneticanalysis of gymnosperms and the safemedication of Ephedra.

Data Availability

The assembled complete CP genome sequences of the threespecies were submitted to NCBI with the accession numbers

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Clade A

Clade B

Clade A1

Clade A2

Ephedra equisetina MH161420Ephedra equisetina NC_011954Ephedra intermedia MH161421Ephedra sinica MH161422Ephedra foeminea KT934791Gnetum montanum KC427271Gnetum parvifolium NC_011942Gnetum ula AP014923Gnetum gnemon KP099649Welwitschia mirabilis AP009568Cephalotaxus wilsoniana NC_016063Pinus koraiensis NC_004677Larix decidua NC_016058Ginkgo biloba AB684440Cycas debaoensis KU743927Cycas taitungensis NC_009618Cycas revoluta NC_020319

Gymnospermae

Equisetum arvense NC_014699Selaginella uncinata AB197035

Pteridophyta

100100

89

100

100100

100

100

100

100

99100

100

99

100

99

99

96/

100/

100/

Figure 7: Phylogenetic trees constructed usingML andMPmethods based on common protein-coding genes of three Ephedra and other 16 species.Numbers (MP/ML) above the branches are bootstrap support values. Only one support value means the same value.

MH161420 (E. equisetina), MH161421 (E. intermedia), andMH161422 (E. sinica). Users could download the data as areference for research purposes only.

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper.

Acknowledgments

The study was supported by CAMS Innovation Fund forMedical Sciences (CIFMS) (No. 2016-I2M-3-016), NationalMajor Scientific andTechnological Special Project for “Signif-icant New Drugs Development” (No. 2018ZX09711001-008-007), and the open topic of Shanghai Key Laboratory of PlantFunctional Genomics and Resources (Shanghai ChenshanBotanical Garden) (No. PFGR201703).

Supplementary Materials

Table S1: validated primers for confirming four boundaries ofthe CP genomes from three Ephedra species. Table S2: thedetails of the selected species in the ML trees. Table S3: thecodon usage of CP genomes of three Ephedra species. TableS4: SSR types and numbers of CP genomes of three Ephedraspecies. (Supplementary Material)

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